Hydrophobic interaction chromatography (HIC) is a purification technique that exploits the interaction of HIC media with hydrophobic regions present on a protein of interest, such as an antibody, and/or those present on an impurity to separate a protein of interest present in a sample mixture. HIC is often utilized in either a bind-elute mode, in which the protein of interest remains bound to HIC media until eluted during an elution phase, or a flow through mode, in which the protein of interest flows through the column while the impurity binds to the media.
Recently, a chromatographic method termed “weak partitioning mode” has been described for the purification of proteins (U.S. Pat. No. 8,067,182). According to U.S. Pat. No. 8,067,182, this method allows for the binding of both product and impurity and is defined by a intermediate partition coefficient (Kp) for the product. Compared to the flow-through mode, in which the Kp for the product is typically low (e.g., <0.1), thereby allowing the product to flow through the column while the impurity is bound, and the bind-elute mode, in which the Kp for the product is typically high (e.g., >20), thereby allowing the product to remain bound until eluted during an elution phase, in the weak portioning mode, the Kp for the product is in the range of 0.1-20.
Importantly, U.S. Pat. No. 8,067,182 teaches the criticality of this Kp range. Specifically, U.S. Pat. No. 8,067,182 teaches that Kp values greater than 20 result in a decreased load challenge at the point of contaminant breakthrough as the product begins to compete with the contaminant for binding sites on the media. In addition, U.S. Pat. No. 8,067,182 teaches that Kp values greater than 20 result in decreased product recovery in that the isocratic wash conditions are not effective at washing the bound product off the column in a reasonable number of wash volumes. Accordingly, U.S. Pat. No. 8,067,182, stresses the criticality of a Kp range to achieve desired purification (see columns 9 and 10).
When applied to HIC, the weak partitioning mode described in U.S. Pat. No. 8,067,182 requires an even more narrow Kp range. As set forth in Example 4, weak partitioning for HIC required a Kp less than 10. Patentees report that HIC performance deteriorates with respect to both contaminant reduction and product recovery at stronger binding conditions.